Classical swine H1N1 influenza viruses confer cross protection from swine-origin 2009 pandemic H1N1 influenza virus infection in mice and ferrets

The hemagglutinin of the 2009 pandemic H1N1 influenza virus is a derivative of and is antigenically related to classical swine but not to seasonal human H1N1 viruses. We compared the A/California/7/2009 (CA/7/09) virus recommended by the WHO as the reference virus for vaccine development, with two classical swine influenza viruses A/swine/Iowa/31 (sw/IA/31) and A/New Jersey/8/1976 (NJ/76) to establish the extent of immunologic cross-reactivity and cross-protection in animal models. Primary infection with 2009 pandemic or NJ/76 viruses elicited antibodies against the CA/7/09 virus and provided complete protection from challenge with this virus in ferrets; the response in mice was variable and conferred partial protection. Although ferrets infected with sw/IA/31 virus developed low titers of cross-neutralizing antibody, they were protected from pulmonary replication of the CA/7/09 virus. The data suggest that prior exposure to antigenically related H1N1 viruses of swine-origin provide some protective immunity against the 2009 pandemic H1N1 virus.     

Natural co-infection of influenza A/H3N2 and A/H1N1pdm09 viruses resulting in a reassortant A/H3N2 virus

BackgroundDespite annual co-circulation of different subtypes of seasonal influenza, co-infections between different viruses are rarely detected. These co-infections can result in the emergence of reassortant progeny.Study designWe document the detection of an influenza co-infection, between influenza A/H3N2 with A/H1N1pdm09 viruses, which occurred in a 3 year old male in Cambodia during April 2014. Both viruses were detected in the patient at relatively high viral loads (as determined by real-time RT-PCR CT values), which is unusual for influenza co-infections. As reassortment can occur between co-infected influenza A strains we isolated plaque purified clonal viral populations from the clinical material of the patient infected with A/H3N2 and A/H1N1pdm09.ResultsComplete genome sequences were completed for 7 clonal viruses to determine if any reassorted viruses were generated during the influenza virus co-infection. Although most of the viral sequences were consistent with wild-type A/H3N2 or A/H1N1pdm09, one reassortant A/H3N2 virus was isolated which contained an A/H1N1pdm09 NS1 gene fragment. The reassortant virus was viable and able to infect cells, as judged by successful passage in MDCK cells, achieving a TCID₅₀ of 10⁴/ml at passage number two. There is no evidence that the reassortant virus was transmitted further. The co-infection occurred during a period when co-circulation of A/H3N2 and A/H1N1pdm09 was detected in Cambodia.ConclusionsIt is unclear how often influenza co-infections occur, but laboratories should consider influenza co-infections during routine surveillance activities.     

A novel monoclonal antibody effective against lethal challenge with swine-lineage and 2009 pandemic H1N1 influenza viruses in mice

The HA protein of the 2009 pandemic H1N1 viruses (H1N1pdm) is antigenically closely related to the HA of classical North American swine H1N1 influenza viruses (cH1N1). Since 1998, through mutation and reassortment of HA genes from human H3N2 and H1N1 influenza viruses, swine influenza strains are undergoing substantial antigenic drift and shift. In this report we describe the development of a novel monoclonal antibody (S-OIV-3B2) that shows high hemagglutination inhibition (HI) and neutralization titers not only against H1N1pdm, but also against representatives of the α, β, and γ clusters of swine-lineage H1 influenza viruses. Mice that received a single intranasal dose of S-OIV-3B2 were protected against lethal challenge with either H1N1pdm or cH1N1 virus. These studies highlight the potential use of S-OIV-3B2 as effective intranasal prophylactic or therapeutic antiviral treatment for swine-lineage H1 influenza virus infections.     

Transmission of influenza A/H5N1 viruses in mammals

Highly pathogenic avian H5N1 influenza A viruses occasionally infect humans and cause severe respiratory disease and fatalities. Currently, these viruses are not efficiently transmitted from person to person, although limited human-to-human transmission may have occurred. Nevertheless, further adaptation of avian H5N1 influenza A viruses to humans and/or reassortment with human influenza A viruses may result in aerosol transmissible viruses with pandemic potential. Although the full range of factors that modulate the transmission and replication of influenza A viruses in humans are not yet known, we are beginning to understand some of the molecular changes that may allow H5N1 influenza A viruses to transmit via aerosols or respiratory droplets among mammals. A better understanding of the biological basis and genetic determinants that confer transmissibility to H5N1 influenza A viruses in mammals is important to enhance our pandemic preparedness.     

Differences in transmissibility and pathogenicity of reassortants between H9N2 and 2009 pandemic H1N1 influenza A viruses from humans and swine

Both H9N2 subtype avian influenza and 2009 pandemic H1N1 viruses (pH1N1) can infect humans and pigs, which provides the opportunity for virus reassortment, leading to the genesis of new strains with potential pandemic risk. In this study, we generated six reassortant H9 viruses in the background of three pH1N1 strains from different hosts (A/California/04/2009 [CA04], A/Swine/Jiangsu/48/2010 [JS48] and A/Swine/Jiangsu/285/2010 [JS285]) by replacing either the HA (H9N1-pH1N1) or both the HA and NA genes (H9N2-pH1N1) from an h9.4.2.5-lineage H9N2 subtype influenza virus, A/Swine/Taizhou/5/08 (TZ5). The reassortant H9 viruses replicated to higher titers in vitro and in vivo and gained both efficient transmissibility in guinea pigs and increased pathogenicity in mice compared with the parental H9N2 virus. In addition, differences in transmissibility and pathogenicity were observed among these reassortant H9 viruses. The H9N2-pH1N1viruses were transmitted more efficiently than the corresponding H9N1-pH1N1 viruses but showed significantly decreased pathogenicity. One of the reassortant H9 viruses that were generated, H9N-JS48, showed the highest virulence in mice and acquired respiratory droplet transmissibility between guinea pigs. These results indicate that coinfection of swine with H9N2 and pH1N1viruses may pose a threat for humans if reassortment occurs, emphasizing the importance of surveillance of these viruses in their natural hosts.     

Genetic diversity of HA1 domain of hemagglutinin gene of pandemic influenza H1N1pdm09 viruses in New Delhi, India

Genetic analysis of pandemic 2009 influenza A (H1N1; H1N1pdm09) virus was undertaken to understand virus evolution during 2009 and 2010 in India. Surveillance of influenza viruses from July 2009 to December 2010 revealed major peaks of circulating H1N1pdm09 viruses in August–September and December–January 2009 and then in August–September 2010. To understand the diversity of the H1N1pdm09 virus, selected specimens (n = 23) from 2009 or 2010 were characterized by nucleotide sequence determination of the HA1 subunit of the HA gene. Phylogenetic analysis revealed that 22 clustered with clade 7 viruses characterized by S203T mutations, whereas one virus from 2010 fell within clade 6. None of the viruses from either 2009 or 2010 formed a monophyletic group, suggesting a continuum of independent introduction of circulating viral strains. Amino acid analysis revealed minor amino acid changes in the antigenic or receptor‐binding domains. Importantly, we observed mutations that were also present in 1918 pandemic virus, which includes S183P in 4 and S185T mutation in 3 of 13 viruses analyzed from 2010, while none of the 2009 viruses carried these mutations. Whether antibody‐mediated pressure is imposing such changes remains to be determined. Continued genetic surveillance is warranted to monitor pathogenicity as the virus evolves to acquire new features. J. Med. Virol. 84:386–393, 2012. © 2011 Wiley Periodicals, Inc.     

Emergence of novel reassortant H3N2 swine influenza viruses with the 2009 pandemic H1N1 genes in the United States

Reassortant H1 swine influenza viruses (SIVs) carrying 2009 pandemic H1N1 virus (pH1N1) genes have been isolated from pigs worldwide. Seven novel reassortant H3N2 SIVs were identified from diseased pigs in the USA from winter 2010 to spring 2011. These novel viruses contain three or five internal genes from pH1N1 and continue to circulate in swine herds. The emergence of novel reassortant H3N2 SIVs demonstrates reassortment between pH1N1 and endemic SIVs in pigs and justifies continuous surveillance.     

Characterization of an influenza A virus in Mexican swine that is related to the A/H1N1/2009 pandemic clade

In the spring of 2009, swine-origin influenza H1N1pdm09 viruses caused the first influenza pandemic of this century. We characterized the influenza viruses that circulated early during the outbreak in Mexico, including one newly sequenced swine H1N1pdm09 virus and three newly sequenced human H1N1pdm09 viruses that circulated in the outbreak of respiratory disease in La Gloria, Veracruz. Phylogenetic analysis revealed that the swine isolate (A/swine/Mexico/4/2009) collected in April 2009 is positioned in a branch that is basal to the rest of the H1N1pdm09 clade in two (NP and PA) of the eight single-gene trees. In addition, the concatenated HA-NA and the complete whole-genome trees also showed a basal position for A/swine/Mexico/4/2009. Furthermore, this swine virus was found to share molecular traits with non-H1N1pdm09 H1N1 viral lineages. These results suggest that this isolate could potentially be the first one detected from a sister lineage closely related to the H1N1pdm09 viruses.     

Isolation and characterization of two H5N1 influenza viruses from swine in Jiangsu Province of China

Pigs are susceptible to infection with both human and avian influenza A viruses and are considered intermediate hosts that facilitate virus reassortment. Although H5N1 virus has spread to a wide range of avian and mammalian species, data about swine H5N1 isolates are scarce. To determine whether Asian H5N1 influenza viruses had been transmitted to pigs, a total of 1,107 nasal swab samples from healthy swine were collected from 2008 to 2009 in Jiangsu province of eastern China. In this survey, two H5N1 viruses A/swine/Jiangsu/1/2008 (JS/08) and A/swine/Jiangsu/2/2009 (JS/09) were isolated and identified. Phylogenetic analysis showed that JS/08 and JS/09 belonged to clade 7 and clade 2.3.4, respectively, and shared over 99.0 % sequence identity with poultry H5N1 isolates of the same clade in China. Receptor specificity analysis also showed that both of the swine H5N1 isolates bound preferentially to avian-type receptors. However, experiments in mammals indicated that JS/09 was moderately pathogenic to mice without prior adaption, whereas JS/08 had limited ability to replicate. Our findings suggest that pigs are naturally infected with avian H5N1 virus and highlight the potential threat to public health due to adaption or reassortment of H5N1 virus in this species.     

Validation of commercial real-time RT-PCR kits for detection of influenza A viruses in porcine samples and differentiation of pandemic (H1N1) 2009 virus in pigs

Swine influenza, apart from its importance in animal health, may also be of public health significance. Although the first human infections with the multi-reassortant H1N1 virus (pH1N1/09) responsible for the 2009 pandemic were not related to pig exposure, this virus was shown to be related genetically to swine influenza viruses (SIV) and easily transmissible to pigs. In addition to direct animal health concerns, transmission and possible adaptation of the pH1N1/09 virus in pigs may have serious consequences on the risk of human infection by increasing the reservoir of this virus and the risk of possible emergence of new reassortant viruses with increased virulence for pigs and/or humans. Sensitive tools to monitor and detect rapidly such an infection are therefore mandatory. In this study, five commercial real-time RT-PCR assays developed by manufacturers LSI and Adiagène were assessed and validated, (i) for rapid detection of influenza A viruses, including pH1N1/09, in pig and (ii) for the differentiation of pH1N1/09 in that species. Two kits target the influenza A virus M gene, two others amplify the pH1N1/09 virus H1 gene and one kit targets the pH1N1/09 virus N1 gene. All five kits are ready-to-use, one-step duplex RT-PCR and contain an internal positive control (IPC), appropriate for porcine biological samples, for assessing RNA extraction efficiency and the presence of PCR inhibitors. They have been used successfully by veterinary laboratories and shown to be powerful tools for the diagnosis and epidemiological surveillance of influenza virus infections in pigs.     

Factors influencing helper-independent adeno-associated virus replication

The 2009 influenza A virus (IAV) pandemic resulted from reassortment of avian, human and swine strains probably in pigs. To elucidate the role of viral genes in host adaptation regarding innate immune responses, we focussed on the effect of genes from an avian H5N1 and a porcine H1N1 IAV on infectivity and activation of porcine GM-CSF-induced dendritic cells (DC). The highest interferon type I responses were achieved by the porcine virus reassortant containing the avian polymerase gene PB2. This finding was not due to differential tropism since all viruses infected DC equally. All viruses equally induced MHC class II, but porcine H1N1 expressing the avian viral PB2 induced more prominent nuclear NF-κB translocation compared to its parent IAV. The enhanced activation of DC may be detrimental or beneficial. An over-stimulation of innate responses could result in either pronounced tissue damage or increased resistance against IAV reassortants carrying avian PB2.     

Restricted infectivity of a human-Lineage H3N2 influenza A virus in pigs is hemagglutinin and neuraminidase gene dependent

Influenza A viruses cause pandemics at sporadic intervals. Pandemic viruses can potentially be introduced into the human population through in toto transfer of an avian influenza virus or through reassortment between avian and human strains. Pigs are believed to play a central role in the creation of pandemic viruses through reassortment because of their susceptibility to infection with both avian and human influenza viruses. However, we recently found that a human-lineage H3N2 influenza virus was highly restricted in its ability to infect pigs after intranasal inoculation. We hypothesized that this restricted infectivity phenotype was controlled by the hemagglutinin (HA) and neuraminidase (NA). To test this, we infected pigs with reverse genetics-created HA plus NA reassortant viruses. Specifically, introduction of the HA and NA genes of a contemporary H3N2 swine virus into the genetic background of the wholly human virus resulted in a significant increase in virus shedding and pathogenicity. These data indicate that the HA/NA can play important roles in controlling human influenza virus infectivity in pigs. The results further support the premise that a barrier exists to human influenza virus infection in pigs, which may limit the role of pigs in pandemic virus creation through reassortment of human and avian influenza viruses.     

Characterization of oseltamivir‐resistant influenza A(H1N1)pdm09 viruses in Taiwan in 2009–2011

The early isolated swine‐origin influenza A(H1N1)pdm09 viruses were susceptible to oseltamivir; however, there is a concern about whether oseltamivir‐resistant influenza A(H1N1)pdm09 viruses will spread worldwide as did the oseltamivir‐resistant seasonal influenza A(H1N1) viruses in 2007–2008. In this study, the frequency of oseltamivir resistance in influenza A(H1N1)pdm09 viruses was determined in Taiwan. From May 2009 to April 2011, 1,335 A(H1N1)pdm09‐positive cases in Taiwan were tested for the H275Y mutation in the neuraminidase (NA) gene that confers resistance to oseltamivir. Among these, 15 patients (1.1%) were found to be infected with H275Y virus. All the resistant viruses were detected after the patients have received the oseltamivir. The overall monthly ratio of H275Y‐harboring viruses ranged between 0% and 2.88%, and the peak was correlated with influenza epidemics. The genetic analysis revealed that the oseltamivir‐resistant A(H1N1)pdm09 viruses can emerged from different variants with a great diversity under drug pressure. The ratio of NA/HA activities in different clades of oseltamivir‐resistant viruses was reduced compared to those in the wild‐type viruses, indicating that the balance of NA/HA in the current oseltamivir‐resistant influenza A(H1N1)pdm09 viruses was interfered. It is possible that H275Y‐bearing A(H1N1)pdm09 virus has not yet spread globally because it lacks the essential permissive mutations that can compensate for the negative impact on fitness by the H275Y amino acid substitution in NA. Continuous monitoring the evolution patterns of sensitive and resistant viruses is required to respond to possible emergence of resistant viruses with permissive genetic background which enable the wide spread of resistance. J. Med. Virol. 85:379–387, 2013. © 2012 Wiley Periodicals, Inc.     

Genetic evolution of swine influenza A (H3N2) viruses in China from 1970 to 2006

Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts, or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of wholly human-like H3N2 viruses, double-reassortant H3N2 viruses, and triple-reassortant H3N2 viruses in pigs in China by analyzing the eight genes of swine influenza A (H3N2) viruses found in China from 1970 to 2006. In 1970, the first wholly human-like H3N2 (Hong Kong/68-like) viruses were isolated from pigs in Taiwan, and then in the next years Victoria/75-like, Sydney/97-like, New York/99-like, and Moscow/99-like swine H3N2 viruses were regularly isolated in China. In the 1980s, two triple-reassortant viruses were isolated from pigs. Recently, the double-reassortant viruses containing genes from the human (HA and NA) and avian (PB2, PB1, PA, NP, M, and NS) lineages and the triple-reassortant viruses containing genes from the human (HA and NA), classical swine (NP), and avian (PB2, PB1, PA, M, and NS) lineages emerged in pigs in China. The coexistence of wholly human-like and reassortant viruses provides further evidence that pigs serve as intermediate hosts, or mixing vessels, and emphasizes the importance of reinforcing swine influenza virus surveillance in China.     

Genetic analysis and antigenic characterization of swine origin influenza viruses isolated from humans in the United States, 1990-2010

Swine influenza viruses (SIV) have been recognized as important pathogens for pigs and occasional human infections with swine origin influenza viruses (SOIV) have been reported. Between1990 and 2010, a total of twenty seven human cases of SOIV infections have been identified in the United States. Six viruses isolated from1990 to 1995 were recognized as classical SOIV (cSOIV) A(H1N1). After 1998, twenty-one SOIV recovered from human cases were characterized as triple reassortant (tr_SOIV) inheriting genes from classical swine, avian and human influenza viruses. Of those twenty-one tr_SOIV, thirteen were of A(H1N1), one of A(H1N2), and seven of A(H3N2) subtype. SOIV characterized were antigenically and genetically closely related to the subtypes of influenza viruses circulating in pigs but distinct from contemporary influenza viruses circulating in humans. The diversity of subtypes and genetic lineages in SOIV cases highlights the importance of continued surveillance at the animal-human interface.     

The emergence of novel swine influenza viruses in North America

Since 1997, novel viruses of three different subtypes and five different genotypes have emerged as agents of influenza among pigs in North America. The appearance of these viruses is remarkable because there were no substantial changes in the overall epidemiology of swine influenza in the United States and Canada for over 60 years prior to this time. Viruses of the classical H1N1 lineage were virtually the exclusive cause of swine influenza from the time of their initial isolation in 1930 through 1998. Antigenic drift variants of these H1N1 viruses were isolated in 1991-1998, but a much more dramatic antigenic shift occurred with the emergence of H3N2 viruses in 1997-1998. In particular, H3N2 viruses with genes derived from human, swine and avian viruses have become a major cause of swine influenza in North America. In addition, H1N2 viruses that resulted from reassortment between the triple reassortant H3N2 viruses and classical H1N1 swine viruses have been isolated subsequently from pigs in at least six states. Finally, avian H4N6 viruses crossed the species barrier to infect pigs in Canada in 1999. Fortunately, these H4N6 viruses have not been isolated beyond their initial farm of origin. If these viruses spread more widely, they will represent another antigenic shift for our swine population, and could pose a threat to the world's human population. Research on these novel viruses may offer important clues to the genetic basis for interspecies transmission of influenza viruses.     

Molecular characterization of the influenza A(H1N1)pdm09 isolates collected in the 2015-2016 season and comparison of HA mutations detected in Turkey since 2009

Influenza A(H1N1)pdm09 pandemic virus causing the 2009 global outbreak moved into the post-pandemic period, but its variants continued to be the prevailing subtype in the 2015-2016 influenza season in Europe and Asia. To determine the molecular characteristics of influenza A(H1N1)pdm09 isolates circulating during the 2015-2016 season in Turkey, we identified mutations in the hemagglutinin (HA) genes and investigated the presence of H275Y alteration in the neuraminidase genes in the randomly selected isolates. The comparison of the HA nucleotide sequences revealed a very high homology (>99.5%) among the studied influenza A(H1N1)pdm09 isolates, while a relatively low homology (96.6%-97.2%), was observed between Turkish isolates and the A/California/07/2009 vaccine virus. Overall 14 common mutations were detected in HA sequences of all 2015-2016 influenza A(H1N1)pdm09 isolates with respect to the A/California/07/2009 virus, four of which located in three different antigenic sites. Eleven rare mutations in 12 HA sequences were also detected. Phylogenetic analysis revealed that all characterized influenza A(H1N1)pdm09 isolates formed a single genetic cluster, belonging to the genetic subclade 6B.1, defined by HA amino acid substitutions S84N, S162N, and I216T. Furthermore, all isolates showed an oseltamivir-sensitive genotype, suggesting that Tamiflu (Oseltamivir) could still be the drug of choice in Turkey.     

Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses

The panzootic caused by A/goose/Guangdong/1/96‐lineage highly pathogenic avian influenza (HPAI) A(H5) viruses has occurred in multiple waves since 1996. From 2013 onwards, clade 2.3.4.4 viruses of subtypes A(H5N2), A(H5N6), and A(H5N8) emerged to cause panzootic waves of unprecedented magnitude among avian species accompanied by severe losses to the poultry industry around the world. Clade 2.3.4.4 A(H5) viruses have expanded in distinct geographical and evolutionary pathways likely via long distance migratory bird dispersal onto several continents and by poultry trade among neighboring countries. Coupled with regional circulation, the viruses have evolved further by reassorting with local viruses. As of February 2019, there have been 23 cases of humans infected with clade 2.3.4.4 H5N6 viruses, 16 (70%) of which had fatal outcomes. To date, no HPAI A(H5) virus has caused sustainable human‐to‐human transmission. However, due to the lack of population immunity in humans and ongoing evolution of the virus, there is a continuing risk that clade 2.3.4.4 A(H5) viruses could cause an influenza pandemic if the ability to transmit efficiently among humans was gained. Therefore, multisectoral collaborations among the animal, environmental, and public health sectors are essential to conduct risk assessments and develop countermeasures to prevent disease and to control spread. In this article, we describe an assessment of the likelihood of clade 2.3.4.4 A(H5) viruses gaining human‐to‐human transmissibility and impact on human health should such human‐to‐human transmission occur. This structured analysis assessed properties of the virus, attributes of the human population, and ecology and epidemiology of these viruses in animal hosts.